Problems associated with cancer cell separation from the or tissue of patients with metastatic cancer during traditional bone marrow harvest and leukopheresis procedures have been reported (Campana, D. et al. Detection of Minimal Residual Disease in Acute Leukemia: Methodological Advances and Clinical Significance, Blood, 1995 Mar. 15, 85 (6): 1416-34; Brugger, W. et al., Mobilization of Tumor Cells and Hematopoietic Progenitor Cells into Peripheral Blood of Patients with Solid Tumors, Blood, 83 (3): 636-40, 1994). It is estimated that among the order of 10 billion total mononuclear cells harvested from a patient, there are 25 thousand to 12 million contaminating cancer cells. These contaminating cancer cells have been shown by genetic marking to contribute to relapse (Rill, E R et al., Direct Demonstration that Autologous Bone Marrow Transplantation for Solid Tumors Can Return a Multiplicity of Tumorigenic Cells, Blood, 84 (2): 380-383, 1994).
Large numbers of cancer cells were also found in the circulation of cancer patients with metastatic diseases. Glaves, D., R P Huben, & L. Weiss (1988. Br. J. Cancer. 57: 32-35) took samples of blood from the renal vein in 10 patients just prior to surgery of renal cell carcinoma and estimated that cancer cells were being released at rates of 107 to 109 cells per day. How these circulating cancer cells contribute to metastasis remains unknown. A major stumbling block is the difficulty involved in identifying an extremely minor subpopulation of circulating cancer cells, ranging from one of thousands to millions of cells, which are metastatic. It is apparent that majority of circulating cancer cells are killed due to host immunity. For examples, in experimental animal tumor models where the use of antibody-based cell separation is more reliable, it has been estimated that about 10 to 100 million tumor cells are released into the blood during the growth of transplantable B16 melanomas and Lewis lung tumors (approximately 20 days), however, these cells give rise to less than 100 lung metastases per mouse (Glaves, D., 1983, Br. J. Cancer, 48: 665-673). Furthermore, in the large number of experiments in which tumor cells have been introduced directly into the circulation of mice or rats it is rare that more than 0.01% of such cells form tumor nodules. More commonly the efficiency is two or more orders of magnitude lower. These experimental data suggest that the initial release of cancer cells from the primary tumor is not the limiting factor in metastatic development as only a very small fraction of shed cancer cells are viable, invasive, and, therefore, metastatic. It is essential to develop a cell separation and detection system targeting on such metastatic cells for the understanding of mechanism of metastasis.
Several methods are known for separating cancer cells from blood or body fluids. Such methods include, for example, separating cancer cells one by one by microdissection (Suarez-Quian et al., 1999, Biotechniques, 26: 328-35; Beltinger and Debatin, 1998, Mol. Pathol. 51: 233-6) or by antibody-based methods using fluorescence activated cell sorting (Pituch-Noworolska et al., 1998, Int. J. Mol. Med. 1: 573-8), separating cancer cells coated with antibodies on a magnetic material through the use of a magnetic field (Denis et al., 1997, Int. J. Cancer 74: 540-4; Racila et al., 1998, Proc. Natl. Acad Sci USA 95-4589-94), or separating circulating cancer cells on density gradients (Sabile et al., 1999, Am. J. Clin. Pathol. 112: 171-8). However, such cancer cell separation methods are dependent on the availability of tumor specific antibodies or the buoyant density and morphology unique to different cancer cells. Thus, a great need exists for efficient methods for removing cancer cells from a hematopoietic cell transplant (Gulati, S C et al. Rationale for Purging in Autologous Stem Cell Transplantation. Journal of Hematotherapy, 2 (4): 467-71, 1993).
As demonstrated in early studies, primary cancers begin shedding neoplastic cells into the circulation at an early stage of metastases formation (Fidler I J, 1973, European Journal of Cancer 9: 223-227; Liotta L A et al., 1974, Cancer Research 34: 997-1004). Once shed into the circulation, cancer cells adhere to the basement membrane underlying vessel walls and invade adjacent connective tissue leading to formation of micrometastases (Liotta et al., 1991, Cell 64: 327-336). It is postulated that cancer cells present in the invasion front and those shed into the circulation are critically involved in the progression of metastatic diseases.
The metastatic process is complex, involving escape of a cancer cell from the primary tumor, movement to a new location and establishment of growth at the new site. To successfully metastasize, the invasive cancer cells must acquire the following metastatic properties: (i) shedding from primary carcinoma, (ii) survival in the circulation and growth on vessel wall, (iii) the ability to invade (adhere to, and subsequently degrade and ingest) collagenous matrix, and (iv) extravasation, colonization and cooperation with angiogenesis (Chambers et al., 1998, Cancer & Metastasis Review 17: 263-269).
The various steps associated with the process are essentially the same whether the cell escapes into lymphatic or blood vessels, and they involve an essential cellular property, i.e., cell invasiveness. Cancer invasiveness requires the adhesion to, and the degradation and ingestion of the extracellular matrix (ECM) by invading cancer cells, accompanied by translocation or migration of the cells into the ECM. Such cellular activities occur on membrane protrusions referred to as invadopodia, which exhibit dynamic membrane mobility, ECM adhesion, and degradation. Recent evidence has demonstrated the involvement of serine integral membrane proteases (SIMP), including dipetidyl peptidase IV (DPPIV)/CD26 and seprase, in cell surface proteolysis (Chen, W-T, 1996, Enzyme Protein 49: 59-71).
SIMP members are type II transmembrane proteins comprising cytoplasmic tails that contain 6 amino acids followed by a 20 (seprase) or 22 (DPPIV) amino acid transmembrane domain at the N-terminus and a stretch of 200 amino acids at the C-terminus that constitutes a catalytic region with the catalytic serine in a non-classical orientation (Goldstein, L A et al., 1997, Biochem. Biophys. Acta. 1361:11-19). DPPIV specifically removes N-terminal dipeptides from oligo-peptides with either L-proline, L-hydroxyproline, or L-alanine at the penultimate position. Such peptides include Neuro-Peptide Y and other peptide hormones (Heins, J et al., 1988, Biochim. Biophys. Acta 954: 161-169; Walter, R et al., 1980, Mol. Cell Biochem. 30: 111-126). In addition, a recent report showed that DPPIV also possesses a seprase-like gelatinase and endopeptidase activity, suggesting its involvement in collagen degradation (Bermpohl F et al., 1998, FEBS Letters 428: 152-156). In addition, DPPIV is expressed constitutively on brush border membranes of intestine and kidney epithelial cells (Yaron and Naider, F, 1993, Crit. Rev. Biochem. Mol. Biol. 28: 31-81; Morimoto C. and Schlossman S F, 1994, Immunologist 2: 4-7) and transiently expressed on T-cells implicating DPPVI as a marker for T-cell activation (Morimoto C. and Schlossman S F, 1994, Immunologist 2: 4-7).
Seprase, originally identified as a 170-kDa membrane-bound gelatinase is expressed on invadopodia of highly aggressive melanoma LOX (Aoyama A. and Chen, W. T., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 8296-8300; Mueller, S C et al., 1999, J. Biol. Chem. 274: 24947-24952; Monsky, W L et al., 1994, Cancer Res. 54: 5702-5710). The active enzyme was isolated from cell membranes and shed vesicles of LOX cells. Seprase is a homodimer of 97-kDa subunits (Pineiro-Sanchez, M L et al., 1997, J. Biol. Chem. 272: 7595-7601). Analysis of the deduced amino acid sequence derived from a cDNA that encodes the 97-kDa subunit reveals that the 97-kDa subunit is homologous to DPPIV, and is essentially identical to fibroblast activation protein a (FAPa) (Goldstein et al., 1997 Biochem. Biophys. Acta. 1361, 11-19; Scanlon, M. J et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91: 5657-5661). FAP α is expressed on reactive stromal fibroblasts of epithelial cancers and in healing wounds but not in adult tissue (Garin-Chesa, P. et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 7235-7239).
In carcinoma tumors, however, FAPa was not found to be expressed in carcinoma or endothelial cells (Garin-Chesa et al., 1990, Proc. Natl. Acad. Sci. 87: 7235-7239). Seprase and FAPα differ mainly in a stretch of 45 amino acid residues contiguous with the highly conserved motif GXSXG that contains the active site serine (Scanlan et al., 1994, Proc. Natl. Acad. Sci. USA 91: 5657-5661; Goldstein et al., 1997 Biochem. Biophys. Acta. 1361, 11-19). Recently, an alternatively spliced seprase mRNA was identified from the human melanoma cell line LOX that encodes a novel truncated 27-kDa seprase isoform, that precisely overlaps the carboxyl-terminal catalytic region of 97-kDa seprase (Goldstein and Chen, 2000 J. Biol. Chem. 275: 2554-2559). The splice variant mRNA is generated by an out-of-frame deletion of a 1223-base pair exonic region that encodes part of the cytoplasmic tail, transmembrane, and the membrane proximal-central regions of the extracellular domain (Val(5) through Ser(412)) of the seprase 97-kDa subunit. It is possible that seprase exhibits both gelatinase and Gly-Pro-dipeptidase activities, while the truncated seprase only has the latter dipeptidase activity.
It has long been believed that collagen remodeling is mediated by matrix metalloproteinases (MMP). However, trials with MMP inhibitors (Marimastat, AG3340) and angiogenic inhibitors (angiostatin and endostafin) in patients with cancer have not produced obvious evidence of anti-metastatic, anti-invasive effects. These data indicate that other enzyme systems are needed to replace MMP at the invasion front of a tumor.
Cancer cell invasiveness in vitro can be a direct indication of a tumor's metastatic potential. Knowledge of the cell's invasive phenotype is important in developing cancer treatments that maximize patient survival and quality of life. It is also important in its use in formulating diagnostic tools for detecting cancer progression and metastasis. Therefore, much effort has focused on measuring cancer cell invasiveness, a characteristic of the metastatic potential of carcinomas.
Invasiveness of a cell is often inferred by its cell surface proteolytic activities that degrade extracellular matrix (ECM) components, and that internalize ECM fragments. In vitro assays for such activities are often complicated by other cell surface phenomena such as adhesion, cell surface proteolysis, and membrane mobility. One particular assay designed to measure invasiveness of a cell involves the covalent linkage of fluorescence-labeled or radio labeled fibronectin (or other ECM components) to the surface of a cross-linked gelatin substrata (Chen et al., 1984, J. Cell Biol 98: 1546-1555; Chen, et al., 1985, Nature, 316: 156-158; Chen et al., 1989, J. Exp. Zool. 251: 167-185; Chen et al., 1994, J. Tiss. Cult. Meth. 16: 177-181; Meuller et al., 1989, J. Cell. Biol. 109: 3455-3464). In this particular technique fibronectin was labeled and used to coat over-fixed protein film. The film was then used to measure cell surface proteolytic activities as well as the cellular invasive phenotype in terms of foci of invadopodial extensions and surface indentations in the film. However, this fibronectin-gelatin film assay is of limited value because (i) it uses conventional, over-fixed protein films; (ii) it lacks sensitivity for detecting proteolytic activity of moderately invasive cells such as most tumor cell lines in culture, fibroblasts and angiogenic endothelial cells, (iii) cross-linked gelatin fragments are not ingested by cancer cells; and (iv) it is difficult to build a three dimensional culture gel system from fibronectin and cross-linked gelatin materials. Thus, reliable procedures to measure the invasiveness of such cells will have significant impact in both clinical diagnostic and therapeutic applications of cancer.
The present invention provides a unique, functional based cell separation method to isolate various forms of cancer cells from blood, ascites and primary tumor tissue of patients with metastases, and peripheral blood mononuclear cells including endothelial cells from blood of normal donors. Additionally, the present invention provides evidence that seprase and/or DPPIV are selectively induced in invasive carcinoma cells and in activated fibroblasts (or other tissue cells) and sprouting endothelial cells of malignant tissues thereby providing targets for development of drugs for inhibiting tumor invasion and metastasis.